Citation Manager Formats

Share

Abstract

Objective To investigate whether chronic traumatic encephalopathy (CTE) and CTE with amyotrophic lateral sclerosis (CTE-ALS) exhibit features previously observed in other tauopathies of pathologic phosphorylation of microtubule-associated protein tau at Thr175 (pThr175 tau) and Thr231 (pThr231 tau), and glycogen synthase kinase–3β (GSK3β) activation, and whether these pathologic features are a consequence of traumatic brain injury (TBI).

Methods Tau isoform expression was assayed by western blot in 6 stage III CTE cases. We also used immunohistochemistry to analyze 5 cases each of CTE, CTE-ALS, and 5 controls for expression of activated GSK3β, pThr175 tau, pThr231 tau, and oligomerized tau within spinal cord tissue and hippocampus. Using a rat model of moderate TBI, we assessed tau pathology and phospho-GSK3β expression at 3 months postinjury.

Conclusions Pathologic phosphorylation of tau at Thr175 and Thr231 and activation of GSK3β are features of the tauopathy of CTE and CTE-ALS. These features can be replicated in an animal model of moderate TBI.

Glossary

AD=

Alzheimer disease;

ALS=

amyotrophic lateral sclerosis;

ALSci=

amyotrophic lateral sclerosis with cognitive impairment;

ANOVA=

analysis of variance;

CTE=

chronic traumatic encephalopathy;

GSK3β=

glycogen synthase kinase–3β;

NFT=

neurofibrillary tangle;

pGSK3β=

phospho–glycogen synthase kinase–3β;

pThr175 tau=

tau phosphorylated at threonine 175;

pThr231 tau=

tau phosphorylated at threonine 231;

RT=

room temperature;

TBI=

traumatic brain injury

Chronic traumatic encephalopathy (CTE) is a fatal neurodegenerative disease that is closely associated with traumatic brain injury (TBI).1 Although TBI is typically associated with elite athletes, it also occurs as a result of both recreational and non-sport-related accidents.2,3 While there remains some controversy, a relationship between TBI and amyotrophic lateral sclerosis (ALS) has been observed, with between 4% and 6% of patients with CTE demonstrating either clinical or neuropathologic features consistent with a diagnosis of ALS.4,–,7 CTE is an affective, behavioral, or cognitive disorder associated with repetitive head trauma that has a characteristic pattern of widespread neuronal and glial microtubule-associated tau (tau protein) deposition.8 In patients with concomitant ALS (CTE-ALS), motor neuron degeneration is also observed, including the presence of TDP-43 neuronal cytoplasmic inclusions.9

The presence of a tauopathy in CTE and CTE-ALS raises the potential of a shared pathophysiology with ALS with cognitive impairment (ALSci), where tau is pathologically phosphorylated at threonine 175 (pThr175 tau) and threonine 231 (pThr231 tau), yielding pathogenic tau oligomers that subsequently assemble into insoluble pathologic tau fibrils.1,10,–,15 In this process, pThr175 tau induces phosphorylation and activation of glycogen synthase kinase–3β (GSK3β), which in turn promotes tau phosphorylation at Thr231.16 Supporting the key role of pThr175, pThr175 tau has only been observed in pathologic states, including a broad range of tauopathies.10,13,17

To date, there have been no detailed studies of the phosphorylation state of tau in either CTE or CTE-ALS. Given this, we examined postmortem archival tissues from patients with CTE and patients with CTE-ALS for pThr175, pThr 231, the active isoform of GSK3β (phospho-GSK3β [pGSK3β]), and tau oligomerization. We also examined whether this process can be triggered by moderate TBI in a rodent.

Methods

Standard protocol approvals, registrations, and patient consents

All studies on human tissues were conducted in accordance with the institutional ethics board standards at University Hospital (London, Canada) and Boston University School of Medicine (Massachusetts). All animal protocols were approved by the University of Western Ontario Animal Care Committee in accordance with the Canadian Council on Animal Care.

CTE and CTE-ALS studies

Microscope slides with 6-μm-thick hippocampal and spinal cord sections from neuropathologically confirmed cases of CTE (n = 5) and CTE-ALS (n = 5) in addition to 5 control cases with no neuropathologic evidence of a neurodegenerative disease state (cases 7–19) were used for immunohistochemical studies. An additional 6 cases (cases 1–6) were obtained as frozen tissue from anterior cingulate and temporal pole. All tissue and slides were obtained from the Veterans Affairs–Boston University–Concussion Legacy Foundation brain bank and were neuropathologically diagnosed as stage III CTE.8,18 Demographic data are summarized in table e-1 (http://links.lww.com/WNL/A99).

Tau fractionation and western blot

Tau protein was isolated from the anterior cingulate gyrus and the temporal pole of 6 stage III CTE cases and a single Alzheimer disease (AD) case as a control. Tau isolation, fractionation, dephosphorylation, and western blot were conducted as previously reported (e-Methods, http://links.lww.com/WNL/A100).19,20

Antigen

Antibody complex was visualized with horseradish peroxidase according to the manufacturer's instructions (Vectastain ABC kit, Vector Laboratories, Burlingame, CA), followed by substrate development with 3,3′-diaminobenzidine (DAB). Counterstaining was performed using Harris hematoxylin.

The extent of pathology was described semiquantitatively as previously reported using visualization with a 20× objective under light microscopy (Olympus BX45; Center Valley, PA).10,21 The semiquantitative scale was applied as follows: − = none; ± = fewer than 5; + = fewer than 10; ++ = more than 20 with scattered distribution; +++ = more than 20 but with locally dense distribution; ++++ = more than 20 with a diffuse distribution as observed for each 20× objective field. In addition, the case positive ratio was defined for each antibody as the number of cases showing any pathology (± or more) compared to the total number of cases stained. Spinal cord pathology was assessed by a binary scale due to the sparse nature of pathology where + = pathology present and − = pathology absent.

In vivo studies

Twelve adult female Sprague-Dawley rats were subjected to a 5-mm-diameter craniotomy followed by a single moderate head trauma (3.5 m/s, 2 mm deep, dwell time of 500 ms) using a cortical impactor (Precision Systems [Horsham, PA] model TBI 0310) (moderate TBI). After 3 months, all rats were killed by transcardiac perfusion with ice-cold saline after intraperitoneal injection with a lethal dose of Euthanyl. Six brains were drop-fixed in ice cold Bouin fixative (Thermo-Fisher) for immunohistochemical analysis while 6 were frozen on dry ice for neurochemical analysis. Bouin fixative was used to reduce artefactual tau pathology.22,23 After 24 hours of fixation, tissue was embedded in paraffin.

Western blots

Immunoblots were also performed using isolates from 6 moderate TBI rats and 4 age-matched controls (e-Methods, links.lww.com/WNL/A100). Briefly, brain tissue was homogenized in RIPA buffer containing protease and phosphatase inhibitors followed by concentration determination by modified Bradford assay (Bio-Rad, Hercules, CA). After immunoprecipitation of brain lysate for total tau (T46 antibody), the entire immunoprecipitation yield was run as a western blot (e-Methods). After transfer to nitrocellulose membrane, blots were probed with rabbit anti-pThr175 tau. Gels were stripped for 30 minutes at 50°C and reprobed with rabbit anti–total tau (Abcam, Cambridge, UK). pGSK3β studies were performed on total brain lysate with mouse anti-GSK3β pTyr216 followed by reprobing with mouse anti-total GSK3β (BD Biosciences). Blots were visualized digitally by enhanced chemiluminescence (Perkin-Elmer) (Bio-Rad Chemidoc MP imaging system and acquired with ImageLab 5.2.1 software). Densitometry was conducted in ImageJ.

Immunohistochemistry

Six moderate TBI and 3 age-matched control rat brains were cut to 5–6 μm thickness and stained for pThr175 tau, pThr231 tau, and pTyr216 GSK3β using the same antibodies and protocol used in human cases.

Statistical analysis

Statistical analyses were conducted using SigmaPlot 10.0 software. A one-way analysis of variance (ANOVA) was conducted following a Shapiro-Wilk test for normality. Post hoc Tukey test was conducted and a p value of 0.05 or lower was considered significant.

Results

Western blot of human CTE

Insoluble tau protein isolated from CTE cases contained all 6 isoforms in both phosphorylated and dephosphorylated isolates. This was in contrast to the 3 isoforms constituting the paired helical filament motif in the insoluble fraction observed in a control AD case (figure 1).20

Representative Western blot of chronic traumatic encephalopathy (CTE)–derived fractionated tau protein shows all 6 tau isoforms in the insoluble fraction in distinction to the 3-isoform, paired helical filament (PHF) motif observed in Alzheimer disease (AD). (Probed with mouse anti-T14/T46 total tau antibody; blots shown are from the same case on 2 separate blots.)

Immunohistochemistry in CTE cases

pThr175, pThr231, and T22 immunoreactivity was observed in the hippocampal formation in all cases of CTE and CTE-ALS (table 1, figure 2A). This included tau-immunoreactive neurofibrillary tangles (NFTs) and dystrophic neurites throughout the CA1–4 regions and extending into the entorhinal cortex in all cases. Consistent with the limited pThr175 tau non-neuronal pathology observed previously,10 oligodendroglial tau immunoreactivity was observed to a limited extent in 5 cases only.10 When present in control cases (3/5), tau immunoreactivity was observed only as faint immunoreactive punctate neuronal staining in the absence of NFTs (figure 2B). No neuritic pathology was observed in controls.

We also observed pathologic tau deposition within the spinal cord regardless of the tau phosphoepitope studied (figure 2B). The presence of tau pathology was independent of whether the underlying pathologic diagnosis was CTE or CTE-ALS (table 1) and consisted of sparsely distributed NFTs in motor neurons and dystrophic neurites. In all cases, pathologic inclusions were minimal in number relative to a more diffuse immunoreactivity to pThr231 tau and pThr175 tau. No pThr175 tau staining was observed in control motor neurons, whereas pThr231 tau was observed, but when present, only as diffuse perikaryal staining of motor neurons. Lipofuscin staining was observed in some controls.

We observed a shift in the pattern of immunoreactivity of pGSK3β from primarily nuclear as observed in the control cases to a diffusely cytosolic pattern (figure e-1, http://links.lww.com/WNL/A98). This pattern was observed in hippocampal CA2 and spinal cord motor neurons in all CTE cases while only being present in occasional isolated cells in controls.

Double-immunolabeling of the hippocampus for both pThr175 tau and pThr231 tau consistently demonstrated colocalization of immunoreactivity to pThr175 with pThr231 immunoreactivity. However, the converse was not observed in that not all pThr231 tau immunoreactive hippocampal neurons were also pThr175-positive, suggesting that only a subset of pThr231 tau pathology is also pThr175-positive.10 This is consistent with our previous observations.10 We also observed colocalization of pThr231 tau and T22 (oligomerized tau), suggesting that oligomeric tau was a component of pThr231 tau pathology. Due to the nature of the antibodies, it was not possible to test for colocalization of pThr175 with T22 as they were raised in the same species, not purified and not compatible with the primary antibody labeling system available to us. While we can therefore only infer that pThr175 tau protein colocalizes with oligomeric tau as well, this inference is supported by our previous studies of a range of tauopathies in which serial sections were available for analysis.10 The inference is further supported by our finding that pThr175 tau immunoreactivity consistently colocalized with active GSK3β (figure 3).

pThr175 and pThr231 expression in moderate TBI

Both pThr175 and pThr231 tau neuronal immunoreactivity was observed in moderate TBI rat brains (figure 4). Of note, pThr175-positive neuronal staining was observed in regions distant to the injury site mainly as axonal staining; however, no fibrillar inclusion-type pathology was observed in regions distant from the injury site. While pThr175 tau expression was elevated within the hemisphere ipsilateral to the injury, pThr175 tau expression in the contralateral hemisphere, while appearing to be increased, was not different from controls when normalized against total tau (p = 0.05 and 0.065, respectively, one-way ANOVA with p = 0.008 and F = 5.757). Diffuse pThr231 staining was observed in healthy neurons. In contrast, pThr231 tau immunoreactive pathology was only observed near the site of injury.

We investigated tau protein phosphorylation and GSK3β activation by western blot (figure 4C). pTyr216 GSK3β was elevated in ipsilateral and contralateral hemispheres relative to uninjured controls (p = 0.005 and 0.001, respectively, one-way ANOVA with p < 0.001 and F = 13.928) when normalized against total GSK3β (figure 4). Finally, we observed the same change in localization of pGSK3β in moderate TBI rat brains as in CTE cases. This was quantified by blinded counts in which we observed an increase in diffuse expression of pTyr216 GSK3β in the injured hemisphere compared to uninjured control rats (p = 0.01 by Tukey post hoc test after one-way ANOVA p = 0.004 and F = 7.559; figure e-2, http://links.lww.com/WNL/A98).

Discussion

We have observed that both CTE and CTE-ALS are tauopathies in which pathologic tau aggregates contain aberrantly phosphorylated tau with immunoreactivity to both pThr175 tau and pThr231 tau. The presence of T22 immunoreactivity (recognizing oligomeric tau) is consistent with a pivotal role for phosphorylation at Thr175 in the pathogenesis of CTE and CTE-ALS. The inference can be made on the basis of our previous study, which showed that pThr175 tau pathology only occurs in pathologic conditions and that the expression of pThr175 tau coincides with oligomerized tau in the same neuronal populations, as does the presence of pThr231.10 Of note, this pathologic process of tau phosphorylation can be triggered experimentally in an in vivo model of moderate TBI and is consistent with a previously reported tauopathy induced by repeated trauma in a murine model of TBI, although this latter study did not investigate the physicochemical properties of the tauopathy.24 The finding that the tauopathy of CTE and CTE-ALS consists of the expression of all 6 tau isoforms in both the soluble and insoluble tau isolates confirms that this process is biochemically distinct from the tauopathy of AD.

Consistent with our previous reports, pThr175 tau staining was only observed when other pathologic tau markers were also present10 and as such, pThr175 tau-positive staining in controls was restricted to those individuals with advanced age, where some tau pathology is expected in the hippocampal formation.25 Therefore, pThr175 tau appears to also be an indicator of toxicity and neuronal damage across a broad range of tauopathies and should be considered in future studies as a CSF or blood biomarker in the diagnosis of CTE26 either alone or in combination with other markers of neuronal injury such as 14-3-327 or neurofilament proteins.28

The tau isoform composition profile observed in CTE in this study and previously in ALSci20 in which all 6 tau isoforms are observed in the soluble and insoluble tau isolates is in distinction to that observed in a number of tauopathies, including AD (in which the pathogenic tau marker is the PHF triplet), corticobasal degeneration and progressive supranuclear palsy (4R tauopathies), and Pick disease (3R tauopathy).29,–,31 This could be interpreted as indicative that the tauopathy of CTE, CTE-ALS, and ALSci is a secondary event triggered in response to a primary neuronal injury. In both CTE and CTE-ALS, this can be postulated to be directly due to the TBI itself, a hypothesis that is supported by the in vivo moderate TBI experimental paradigm. While the trigger for the tauopathy of ALSci remains unknown, it is clear that once initiated, the presence of pThr175 leads to a cascade of events that culminates in neuronal death in vitro.16,32

In relationship to our understanding of the pathophysiology of ALS, 4%–6% of patients with CTE also develop ALS (CTE-ALS),7 a rate much higher than the incidence of ALS (2–3 per 100,000) in the general population.9 Unique to motor neuron degeneration associated with CTE-ALS is tau pathology in the spinal cord. The only other instance of a disseminated tauopathy in association with motor neuron degeneration is that observed in the previously hyperendemic Western Pacific variant of ALS, a variant of ALS recognized to be at the intersection of an environmental insult in an at-risk population.33,34

While we have observed pathologic tau phosphorylation in the spinal cords of both patients with CTE and patients with CTE-ALS in this study, the variability observed warrants investigation on a larger cohort of cases with detailed rostrocaudal analysis to discern whether there is a correlation between motor symptom progression and regional tau deposition. We could not undertake such an analysis given the nature of tissue selection (cervical, thoracic, or lumbar for different cases). However, the absence of tau deposition in the spinal cords of patients with sporadic ALS suggests that the spinal motor neuron tauopathy of CTE-ALS is not an incidental finding or secondary to the primary neuronal injury of ALS. It is possible that tau protein phosphorylation and pathology begins early in the neurodegenerative process in CTE or CTE-ALS, in which case spinal cord pathology would be expected to precede symptom onset in patients who would otherwise develop motor impairment. In addition, given previous reports of TDP-43 deposition in the spinal cord of CTE-ALS,9 a much larger study investigating the correlation of TDP-43-positive neuronal cytoplasmic inclusions is required to bring clarity to the contributing pathologies concomitant to motor neuron death.

In these studies, we have observed pThr175 tau, in conjunction with pThr231 tau, oligomerized tau, and changes in active GSK3β localization consistent with a pathologic tauopathy driven by the aberrant phosphorylation of Thr175 tau in CTE and CTE-ALS. While we have shown that moderate TBI can directly drive this process, understanding how this induces pThr175 is the topic of current studies. Given our previous findings that this pathologic cascade of tau phosphorylation can be fully inhibited, and that this inhibition abolishes pThr175 tau-induced neuronal death,16 our work suggests that both CTE and CTE-ALS may be amenable to pharmacologic inhibition of GSK3β activation.

Author contributions

Alexander J. Moszczynski: contributed to design of all studies, conducted or assisted with all experimental procedures, performed all quantification and interpretation of data, wrote manuscript. Wendy Strong: performed tau fractionation experiments and western blot. Kathy Xu: performed animal surgeries. Ann McKee: contributed tissue to the study, performed pathologic diagnoses, edited manuscript. Arthur Brown: supervised and designed animal studies, edited manuscript. Michael J. Strong: supervised and designed all elements of the studies, edited manuscript.

Study funding

Research supported by an operating grant from the Ontario Neurodegenerative Disease Research Initiative and the Windsor-Essex ALS Society.

Disclosure

The authors report no disclosures relevant to the manuscript. Go to Neurology.org/N for full disclosures.

Footnotes

Go to Neurology.org/N for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.

NOTE: All contributors' disclosures must be entered and current in our database before comments can be posted. Enter and update disclosures at http://submit.neurology.org. Exception: replies to comments concerning an article you originally authored do not require updated disclosures.

Stay timely. Submit only on articles published within the last 8 weeks.

Do not be redundant. Read any comments already posted on the article prior to submission.

200 words maximum.

5 references maximum. Reference 1 must be the article on which you are commenting.

5 authors maximum. Exception: replies can include all original authors of the article.

Submitted comments are subject to editing and editor review prior to posting.

I, the first and corresponding author, verify that I have read the contents of the PUBLISHING AGREEMENT form. *

I, the first and corresponding author, verify my disclosures and those of my co-authors are up to date at http://submit.neurology.org. *

Select only one of the three options below: *

I am an Author of this Work, and the Work was prepared on my own time - not as part of my duties as an employee.

I prepared (or cooperated in the preparation of) the Work as part of my duties as an employee, and the Work is, therefore, a "work made for hire", as defined by the United States Copyright Act of 1976, as amended.

I prepared (or participated in the preparation of) the Work as part of my official duties as an officer or employee of the United States Government.

NOTE: All contributors, besides the first/corresponding author, must complete a separate Disputes & Debates Submission Form and provide via email to the editorial office before comments can be posted.